Ojective 1: Characterize the mechanisms by which eltrombopag evades IFN blockade of c-MPL signaling. To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, we previously showed that IFN, a key proinflammatory cytokine implicated in the destruction of HSPCs in SAA, inhibited TPO signaling in human CD34+ HSPCs cultured in the presence of both cytokines. In contrast, eltrombopag could evade this inhibition in vitro, resulting in improved maintenance of progenitors in clonogenic CFU assays, and long-term repopulating cells in NSG transplantation models compared with TPO-containing cultures (FY16). In FY17-FY18, we sought to characterize the mechanisms by which eltrombopag evades IFN blockade of c-MPL signaling. Because activation of both c-MPL and IFN receptor by their respective ligands induces negative regulatory feedback mechanisms from the SOCS family of proteins, we first measured SOCS expression in CD34+ cells exposed to IFN and TPO or eltrombopag. SOCS expression was equally upregulated by TPO and eltrombopag in the presence of IFN, suggesting an alternative explanation for eltrombopags ability to escape IFN-induced perturbation of TPO signaling. In order to clarify the mechanism of IFN-mediated blockade of TPO signaling from c-MPL, we hypothesized that IFN might impair receptor binding affinity of TPO but not eltrombopag. We used microscale thermophoresis (MST) to assess the effect of IFN on TPO binding to its receptor. We first quantified the TPO:c-MPL binding affinity in the absence of IFN by titration of fluorescently labeled TPO with a soluble fragment of the c-MPL extracellular domain (residues Ser24-Trp491). In agreement with previously reported work, TPO and c-MPL displayed a specific two-site interaction with dissociation constants (KD) of 0.15 0.04 nM and 1,066 126 nM, characterizing a high- and low-affinity binding site, respectively. To investigate the impact of IFN on each binding site of the TPO:c-MPL interaction, we titrated c-MPL into a pre-mixed solution containing both TPO and 100-fold molar excess concentration of IFN. Addition of IFN did not significantly perturb TPO:c-MPL high-affinity binding site, as indicated by a dissociation constant (KD = 0.05 0.01 nM) nearly unchanged relative to the KD measured in the absence of IFN (KD = 0.15 0.04 nM). In contrast, the same molar excess concentration of IFN completely disrupted the low-affinity binding site of the TPO:c-MPL complex (KD, not detectable). To confirm that this effect was specific to IFN, we also challenged the TPO:c-MPL interaction with saturating amounts of other cytokines, including stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (Flt3L). SCF and Flt3L had no detectable impact on either binding site of the TPO:c-MPL interaction. We speculated that IFN might directly and competitively bind to c-MPL, but showed no such binding affinity (Fig. 3C). Together, we inferred from these data that IFN induced a dose-dependent and specific disruption of the TPO:c-MPL low-affinity binding site, but without directly interacting with c-MPL. We next investigated the mechanism by which IFN disrupted the low-affinity binding interaction between TPO and c-MPL. We hypothesized that IFN may bind to TPO, forming heteromeric complexes that sterically hinder the low-affinity TPO:c-MPL binding site, whereas the non-peptidyl small molecule eltrombopag could evade that process. We used MST to investigate TPO:IFN heteromer formation. Notably, these experiments revealed a specific, one-site heteromeric interaction between TPO and fluorescently labeled IFN, with an affinity (KD = 488 76 nM) superior to the TPO:c-MPL low-affinity interaction (KD = 1,066 126 nM). By contrast, no interaction was observed between IFN and other early-acting hematopoietic cytokines (SCF and Flt3L). When TPO was used as the labeled binding partner, we detected a similar high-affinity TPO:IFN interaction (KD = 402 89 nM), and no evidence of complex formation between TPO and SCF or Flt3L. Eltrombopag had no affinity for IFN in the nanomolar to high micromolar concentration range, as demonstrated by MST and fluorescence spectroscopy. Thus, formation of TPO:IFN heteromers appears to represent a previously unrecognized mechanism by which IFN hinders TPO:c-MPL interaction in human HSPCs. Moreover, because eltrombopag cannot complex with IFN, this finding provides an explanation for its ability to overcome IFN-mediated obstruction of TPO cell signaling pathways. This work was presented at the Plenary Session of the American Society of Hematology. Manuscript is under review. Ojective 2: Demonstrate that Eltrombopag has DNA repair activity in human HSPCs To assess DNA repair activity of epag in FA CD34+ HSPCs, a population that is markedly reduced in these patients, CD34+ HSPCs from 6 healthy individuals were subjected to CRISPR/Cas9-induced knockout mutations in FANCA, the most commonly mutated gene in FA. These FA HSPCs were cultured for 24hrs in the presence of early-acting cytokines (SCF and Flt3-L, SF) alone or supplemented with epag (SFE) or TPO (SFT), prior to induction of DSBs by exposure to 2Gy -irradiation (IR). Cells were then cultured for an additional 1, 5 or 24hrs to assess the kinetics of DNA repair, as measured by decreases in H2AX expression, a marker of IR-induced DNA DSBs. Maximal H2AX expression was measured 1hr after IR of FA HSPCs and was similar for all culture conditions (>90% H2AX+ cells), indicating that epag and TPO do not prevent DNA damage. Five hours after IR, most FA HSPCs cultured with epag or TPO had already resolved the IR-induced DNA DSBs, but much higher percentages of H2AX+ cells were still detected in the control SF group. The observed effect was specific to epag and TPO; removal of SCF had no significant impact on DNA repair. By 24hrs after exposure to IR, FA HSPCs cultured with and without epag or TPO had similarly resolved DNA breaks. These findings indicate that epag and TPO increase the kinetics of DNA DSB repair in FA HSPCs. To gain insights into the mechanisms of DNA repair, we inhibited DNA-PK, an essential component of the NHEJ pathway. Addition of a DNA-PK inhibitor (NU7441) had no impact on DNA DSB formation measured at 1 hour or on DNA repair at 24hrs, but completely abrogated the enhanced kinetics of DSB repair observed at 5hrs with epag and TPO. These data indicate that epag and TPO favor the fast-acting DNA-PK dependent classical NHEJ (C-NHEJ) DNA repair mechanism in FA HSPCs, a pathway known to promote genomic stability. In contrast, cells cultured without epag or TPO resolved DSBs using a slower DNA-PK-independent alternative NHEJ (alt-NHEJ) mechanism in FA HSPCs, a pathway known as the primary mediator of genomic instability. Shunting of DSB repair in rapid C-NHEJ with epag or TPO was associated with substantial increase in survival of -irradiated FA HSPCs compared with control (SF) groups. In contrast, when C-NHEJ DNA repair was inhibited with NU7441, the cell survival benefit observed with epag or TPO was abolished. In colony forming unit (CFU) progenitor assays, -irradiated HSPCs cultured with epag or TPO yielded 4-6-fold more CFUs than control SF groups. Importantly, when -irradiated HSPCs were tested in NSG transplantation assays, a 2-fold increase in human cell engraftment was observed in cultures containing epag or TPO compared to controls (p<0.01), suggesting activation of DNA repair activity by these cytokines in cells with long-term repopulating capacity. Overall, our data indicate that epag and TPO enhance DNA DSB repair in HSPCs by promoting the fast-operating C-NHEJ pathway. A phase II clinical trial has been approved to assess safety and efficacy of epag at improving the hematological manifestations of FA. Patient enrollment has begun.